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The antiviral efficacy of ten antisense phosphorodiamidate morpholino oligomers (PMOs) directed against Equine arteritis virus (EAV), a nidovirus belonging to the family Arteriviridae, was evaluated in mammalian (Vero-E6) cells. Peptide-conjugated PMOs (P-PMOs) supplied in cell culture medium at micromolar concentrations were efficiently taken up by Vero-E6 cells and were minimally cytotoxic. The P-PMOs were designed to base pair to RNA sequences involved in different aspects of EAV amplification: genome replication, subgenomic mRNA synthesis, and translation of genome and subgenomic mRNAs. A novel recombinant EAV, expressing green fluorescent protein as part of its replicase polyproteins, was used to facilitate drug screening. A moderate reduction of EAV amplification was observed with relatively high concentrations of P-PMOs designed to anneal to the 3'-terminal regions of the viral genome or antigenome. To determine if the synthesis of subgenomic mRNAs could be specifically reduced, transcription-regulating sequences essential for their production, but not for the production of genomic RNA, were targeted, but these P-PMOs were found to be ineffective at transcription interference. In contrast, all four P-PMOs designed to base pair with targets in the genomic 5' untranslated region markedly reduced virus amplification in a sequence-specific and dose-responsive manner. At concentrations in the low micromolar range, some of the P-PMOs tested completely inhibited virus amplification. In vitro translation assays showed that these P-PMOs were potent inhibitors of translation. The data suggest that these compounds could be useful as reagents for exploring the molecular mechanics of nidovirus translation and have anti-EAV potential at relatively low concentrations.

Antiviral activity of morpholino oligomers designed to block various aspects of Equine arteritis virus amplification in cell culture